Functional Characterization Of PtrWRKY18, PtrWRKY35 And PtrWRKY89 Transcription Factors In Poplar Defense

Plants are susceptible to pathogen infection due to their sessile life style. Complex signaling pathways are evolved in plants to act against pathogen infection and survive from severe diseases. Among them, salicylic acid(SA)-mediated and jasmonic acid/ethylene(JA/ET)-mediated pathway play vital roles in plant defense. SA-mediated signaling pathway is usually triggered by biotrophic pathogens such as Pseudomonas syringae pv. tomato DC3000(PstDC3000), and results in accumulation of reactive oxygen species(ROS), elevated expressing levels of pathogenesis-related genes(PR) and programmed cell death, as well as the activation of the system acquired resistance(SAR) against the pathogen. In contrast, JA/ET-mediated signaling pathway is triggered by necrotrophic fungi such as Botrytis cinerea, and coupled with the expression of plant defensing 1.2(PDF1.2). Increasing evidences indicates that plants usually orchestrate SA- and JA-mediated signaling pathways antagonistically and plenty of transcription factors, such as NAC proteins, MYB proteins and WRKY proteins, are involved in this process. The WRKY transcription factors have been considered to play important roles in plant defense. In Populus, some WRKY genes have been cloned and characterized for their functions against pathogens. However, due to the large size and high gene redundancy of the poplar genome, studying WRKY genes in isolation would provide little information on the biological function of the poplar WRKY gene family in plant defense. Therefore, the analysis of WRKY family based on global genome and transcriptome of Populus is required for identifying the biological functions of WRKY genes, predicting gene redundancy, screening putative defense-related genes and comprehending the transcription regulatory network of WRKY proteins. In this thesis, the main topics are as follows:According to the information of Populus trichocarpa genome in the database PlantTFDB and Phytozome, the gene IDs, DNA sequences and peptide sequences of P. trichocarpa WRKY(PtrWRKY) genes were obtained and named as Ptr WRKY1 through PtrWRKY100 based on their homology with Arabidopsis WRKY genes. Furthermore, according to the number of WRKY domain and the type of zinc-fingers, the 100 Populus WRKY genes were classified into three big group, namely Group I, Group II and Group III. The phylogenetic tree of WRKY proteins showed that members of the three big groups were clustered, respectively, and the Group II, based on the clades, was divided into five subgroups in terms of Subgroup IIa, Subgroup IIb, Subgroup IIc, Subgroup IId and Subgroup IIe. Amino acid sequence alignment of WRKY peptides showed that PtrWRKY99, although clustered in the Group III, contained a Group II-type zinc finger, and thereby was considered as a sole member.Chromosomal distribution of Ptr WRKY genes illustrated that PtrWRKY genes spread across all the linkage groups(LGs) except LG IX. Totally 82 members were mapped to LGs, while the other 18 were located on yet unmapped scaffolds. The distribution of PtrWRKY genes across the LGs seems to be uneven. LG II contains the largest number of 12(13.79%) PtWRKY genes followed by 8(9.20%) on LG XIV. In contrast, only one WRKY gene was found on LG XV. In addition, to investigate the possible relationship between the WRKY genes and potential segmental duplications, we mapped all Ptr WRKY genes to the duplicated blocks. The results indicated that regional duplications might contribute to the expansion of this gene family in the Populus genome. Sequence analysis indicated that the PtrWRKY proteins contained several conserved motifs, for example, a conserved leucine zipper motif, the consensus sequence of the sumoylation motif, the consensus coactivator motif(LxxLL) and repressor motifs(Lx Lx Lx). One or more nuclear localization signals(NLS) were harbored in 77%(77 out of 100) of the PtrWRKY proteins. These NLSs are composed of 3–20 amino acid residues and normally rich in lysine(Lys) or arginine(Arg). In addition, some of the NLSs were located within the WRKY domains, suggesting that this domain may have bifunctionality in these poplar WRKY genes.Analysis of consensus cis-elements on the promoters of PtrWRKY genes indicated that there were plenty of elements involved in response to averse environments and WRKY protein binding DNA sequence(W box) on these promoters, suggesting the biological functions of Ptr WRKY genes in poplar defense and tolerance, and the inter-regulation of PtrWRKY proteins. Furthermore, digital transcriptome analysis of Populus tomentosa treated with SA, MeJA, Marssonina brunnea,low temperature, wounding and high salinity indicated that the expression of 61 out of 100 PtrWRKY genes were influenced by at least one treatment. Quantitative real-time PCR analysis showed that SA was able to induce PtrWRKY60 and PtrWRKY89 expression in the leaves of Populus trichocarpa. Moreover, PtrWRKY60 and PtrWRKY89 might be involved in the early stage of SA signaling transduction.2. According to phylogenetic analysis, PtrWRKY89, belonging to Group III, together with PtrWRKY54 and Ptr WRKY62, are co-orthologous genes of AtWRKY70 in Arabidopsis, suggesting their possible functional redundancy. Promoter analysis showed that PtrWRKY89 was specifically expressed in mature leaves of both Arabidopsis and Populus, but displayed almost no expression in young leaves. Subcellular localization and yeast auto-activation assay showed that PtrWRKY89 was localized exclusively in nucleus and played as a transcription activator.Heterologous overexpression of PtrWRKY89 in Arabidopsis resulted in higher susceptibility to PstDC3000 and B. cinerea compared to wild type plants(WT). Furthermore, in the transgenic Arabidopsis overexpressing PtrWRKY89, both the marker genes of SA-mediated signaling pathway, like PR genes, and the PDF1.2, which is the marker of JA-mediated signaling pathway displayed reduced expression levels compared to WT. Transgenic poplars overexpressing PtrWRKY89 showed increased resistance to Dothiorella gregaria compared to WT. The expression levels of the PR genes in Populus were significantly elevated in the transgenic lines compared to WT, indicating that PtrWRKY89 positively regulated the SA-mediated signaling pathway in poplar. In addition, PtrWRKY89 overexpression in P. tomentosa caused the constitutive expression of PtoWRKY18 and PtoWRKY35. There are four and three W boxes in the promoters of Pto WRKY18 and PtoWRKY35, respectively. Yeast one-hybrid assay demonstrated that PtrWRKY89 bound to the W boxes of PtoWRKY18 and PtoWRKY35. These results indicated that PtrWRKY89 could regulate Pto WRKY18 and Pto WRKY35 in P. tomentosa directly.3. PtrWRKY18 and PtrWRKY35 from P. trichocarpa are the homologue genes of Pto WRKY18 and PtoWRKY35 in P. tomentosa and they have extremely high similarity in both DNA and protein sequence. PtrWRKY18 and PtrWRKY35 belong to Subgroup IIa and contain a putative leucine zipper region at the N terminal, which is similar to their Arabidopsis homolog AtWRKY18, but different from its Arabidopsis counterpart, the putative leucine zipper region in PtrWRKY18 and PtrWRKY35 showed no function in protein interactions as determined by yeast two hybrid assay. Promoter analysis showed that PtrWRKY18 was mainly expressed in the vascular tissues of young leaves and the tender stems while PtrWRKY35 was expressed weakly in the stems. GUS gene driven by the promoters of PtrWRKY18 and Ptr WRKY35 was induced by SA and microbes in the transgenic Arabidopsis. Subcellular localization and yeast auto-activation assay showed that PtrWRKY18 and PtrWRKY35 were both nucleus-localized proteins without transcriptional activation activity. Overexpressing PtrWRKY18 and Ptr WRKY35 in Arabidopsis resulted in reduced resistance to PstDC3000 but enhanced resistance to B. cinerea. The expression levels of PR genes and PDF1.2 were decreased and increased, respectively in the transgenic plants compared to the WT. In Populus, overexpression of Ptr WRKY18 or PtrWRKY35 could strength the resistance to Melampsora medusa, but the knock-out mutants of PtoWRKY18 and PtoWRKY35, which were generated by CRISPER system, showed no significant difference compared to WT. Furthermore, the PR genes in transgenic poplars showed increased expression levels compared to WT, but no difference between the knock-out mutants and WT was detected.In summary, bioinformatics analysis of Populus WRKY family uncovered the phylogenetic relationship of WRKY members, and a SA-induced PtrWRKY89 was identified via transcriptome analysis. Moreover, it was demonstrated that Ptr WRKY89 directly regulated PtrWRKY18 and Ptr WRKY35 to positively modulate SA-mediated signaling pathway in Populus.